Invitrogen™ Image-iT™ LIVE Red and Green Caspase Apoptosis Detection Kits for microscopy

Description

• Detect and evaluate caspase activation, membrane permeability, and cell cycle using Image-iT LIVE red and green poly and caspase-3 and-7 apoptosis detection kits for microscopy, including fluorescence microscopy and high content screening (HCS)
• The Image-iT LIVE red and green caspase detection kits use fluorescent inhibitor of caspases (FLICA) methodology to detect and report caspase activity, a measure of cell apoptosis
• Specifically, the Image-iT LIVE red and green caspase detection kits contain the substrates SR-VAD-FMK and SR-DEVD-FMK (for red fluorescence), or FAM-VAD-FMK and FAM-DEVD-FMK (green fluorescence), for poly caspases and caspase-3/7 detection, respectively
• They also include Hoechst 33342 and propidium iodide stains, enabling simultaneous evaluation of membrane permeability and cell cycle by microscopy and HCS
• The Image-iT LIVE red and green caspase detection kits for microscopy employ a novel approach to detecting active caspases: both assay kits take advantage of a fluorescent inhibitor of caspases (FLICA) methodology, which is an affinity label
• This reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amino acid sequence
• Different amino acid recognition sequences are used to detect different caspases: valine-alanine-aspartic acid (VAD) for poly-caspases (including caspase-1, -3, -4, -5, -6, -7, -8, and -9), and aspartic acid-glutamic acid-valine-aspartic acid (DEVD) for caspase 3/7
• A sulforhodamine (SR) group or carboxyfluorescein (FAM) group is attached as a reporter
• The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety
• The FLICA inhibitor is cell permeant and noncytotoxic
• Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining red or green fluorescent signal is a direct measure of the amount of active caspase present at the time the inhibitor was added
• The approximate excitation and emission peaks of the FLICA, propidium iodide, and Hoechst 33342 reagents are 488⁄ nm/530 nm, 535⁄ nm/6617 nm, and 350 nm/⁄461 nm, respectively
• The detection reagents included within the Image-iT LIVE Red Poly Caspases, Red Caspase-3/7, Green Poly Caspases, and Green Caspase-3/7 Detection kits are SR-VAD-FMK, SR-DEVD-FMK, FAM-VAD-FMK, and FAM-DEVD-FMK, respectively.